Abstract：
The passage number of cells refers to the number of subculturing processes that the cells have undergone. The effect of passage number on morphological and phenotypical characteristics of cells is of great importance. Advanced glycation end products have also been associated with cell functionality and characteristics. Murine monocyte RAW 264.7 cells differentiate into osteoclasts upon receptor activation caused by nuclear factor-kappa-Β ligand (RANKL) treatment. This study aims to identify the role of passage number on intracellular advanced glycation end products (AGEs) formation and osteoclastogenic differentiation of RAW 264.7 cells. Western blotting was performed to check intracellular AGE formation along with fluorometric analysis using a microplate reader. Tartrate-resistant acid phosphatase (TRAP) staining was performed to check osteoclastogenic differentiation, and qPCR was realized to check the responsible mRNA expression. Immunofluorescence was used to check the morphological changes. Intracellular AGE formation was increased with passaging, and the higher passage number inhibited multinucleated osteoclastogenic differentiation. Osteoclastogenic gene expression also showed a reducing trend in higher passages, along with a significant reduction in F-actin ring size and number. Lower passages should be used to avoid the effects of cell subculturing in in vitro osteoclastogenesis study using RAW 264.7 cells.
摘要：
细胞的传代数是指细胞已经经历的传代培养过程的数目。传代数对细胞形态和表型特征的影响非常重要。晚期糖基化终产物也与细胞功能和特征相关。鼠单核细胞RAW264.7细胞在由核因子-κ-Β配体(RANKL)处理引起的受体激活后分化成破骨细胞。本研究旨在确定传代数对RAW264.7细胞胞内晚期糖基化终产物（AGEs）形成和破骨细胞分化的作用。使用酶标仪进行蛋白质印迹以检查细胞内AGE形成以及荧光分析。进行抗酒石酸酸性磷酸酶（TRAP）染色以检查破骨细胞分化，并实现qPCR以检查负责的mRNA表达。免疫荧光检查形态学变化。细胞内AGE形成随传代增加，较高的传代数会抑制多核破骨细胞的分化。成骨基因表达在较高的传代中也显示出降低的趋势，随着F-肌动蛋白环大小和数量的显著减少。在使用RAW264.7细胞的体外破骨细胞发生研究中，应使用较低的传代以避免细胞传代培养的影响。
